PD-1 Peptide Inhibitors

ABSTRACT

This disclosure provides peptides which have a strong affinity for the checkpoint receptor “programmed death 1” (PD-1). These peptides block the interaction of PD-1 with its ligand PD-L1 and can therefore be used for various therapeutic purposes, such as inhibiting the progression of a hyperproliferative disorder, including cancer; treating infectious diseases; enhancing a response to vaccination; treating sepsis; and promoting hair re-pigmentation or lightening of pigmented skin lesions.

This application is a continuation-in-part of Ser. No. 15/705,333 filedon Sep. 15, 2017, which claims priority to and incorporates by referencein its entirety U.S. Ser. No. 62/395,195 filed on Sep. 15, 2016. Ser.No. 15/705,333, Ser. No. 62/395,195, and each reference cited in thisdisclosure are incorporated herein in their entireties.

This application incorporates by reference the contents of a 1.43 kbtext file created on Feb. 27, 2018 and named“00047900252sequencelisting.txt,” which is the sequence listing for thisapplication.

TECHNICAL FIELD

This disclosure relates generally to immunomodulatory peptides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Graph showing saturatable binding of anti-human PD-1 antibody toJurkat cells.

FIG. 2. Graph showing saturatable binding of PD-L1 Fc to Jurkat cells.

FIGS. 3A-B. Graphs showing effect of peptide QP20 on binding of PD-L1 toPD-1. FIG. 3A, MFI; FIG. 3B, normalized mean fluorescence intensity(MFI).

FIGS. 4A-B. Graphs showing effect of peptide HD20 on binding of PD-L1 toPD-1. FIG. 4A, MFI; FIG. 4B, normalized MFI.

FIGS. 5A-B. Graphs showing effect of peptide WQ20 on binding of PD-L1 toPD-1. FIG. 5A, MFI; FIG. 5B, normalized MFI.

FIGS. 6A-B. Graphs showing effect of peptide SQ20 on binding of PD-L1 toPD-1. FIG. 6A, MFI; FIG. 6B, normalized MFI.

FIG. 7A. Graph showing the effect of an anti-human PD-1 antibody on theinteraction between PD-1-expressing Jurkat T cells and PD-L1-expressingCHO cells that results in inhibition of a PD-1 mediated suppression ofluciferase reporter that is under the control of promoter containingIL-2, NFAT, and NF-kB response elements.

FIG. 7B. Graph showing the effect of an anti-human PD-1 antibody on theinteraction between PD-1-expressing Jurkat T cells and PD-L1-expressingCHO cells (data in 7A expressed as fold inhibition).

FIG. 8A. Graph showing that PD-1 peptide inhibitors inhibit, in adose-dependent manner, the interaction between PD-1-expressing Jurkat Tcells and PD-L1-expressing CHO cells, which results in increasedluciferase reporter expression.

FIG. 8B. Graph showing the effect of an anti-human PD-1 antibody on theinteraction between PD-1-expressing Jurkat T cells and PD-L1-expressingCHO cells (data in 8B expressed as fold inhibition).

FIG. 9. Graph showing IL-2 production by peripheral blood mononuclearcells (PBMCs) in a tetanus toxoid recall assay after culture withpeptides QP20, HD20, WQ20, SQ20, or CQ-22.

FIG. 10. Graph showing IL-4 production by PBMCs in a tetanus toxoidrecall assay after culture with peptides QP20, HD20, WQ20, SQ20, orCQ-22.

FIG. 11. Graph showing IL-6 production by PBMCs in a tetanus toxoidrecall assay after culture with peptides QP20, HD20, WQ20, SQ20, orCQ-22.

FIG. 12. Graph showing IL-10 production by PBMCs in a tetanus toxoidrecall assay after culture with peptides QP20, HD20, WQ20, SQ20, orCQ-22.

FIG. 13. Graph showing IL-17a production by PBMCs in a tetanus toxoidrecall assay, after culture with peptides QP20, HD20, WQ20, SQ20, orCQ-22.

FIG. 14. Graph showing IFNγ production by PBMCs in a tetanus toxoidrecall assay, after culture with peptides QP20, HD20, WQ20, SQ20, orCQ-22.

FIG. 15. Graph showing TNFα production by PBMCs in a tetanus toxoidrecall assay, after culture with peptides QP20, HD20, WQ20, SQ20, orCQ-22.

FIG. 16. Graph showing IL-2 production by PBMCs in a tetanus toxoidrecall assay, after culture with various combinations of peptides QP20,HD20, WQ20, and SQ20.

FIG. 17. Graph showing IL-4 production by PBMCs in a tetanus toxoidrecall assay, after culture with various combinations of peptides QP20,HD20, WQ20, and SQ20.

FIG. 18. Graph showing IL-6 production by PBMCs in a tetanus toxoidrecall assay, after culture with various combinations of peptides QP20,HD20, WQ20, and SQ20.

FIG. 19. Graph showing IL-10 production by PBMCs in a tetanus toxoidrecall assay, after stimulation with various combinations of peptidesQP20, HD20, WQ20, and SQ20.

FIG. 20. Graph showing IL-17a production by PBMCs after stimulation withvarious combinations of peptides QP20, HD20, WQ20, and SQ20.

FIG. 21. Graph showing IFNγ production by PBMCs after culture withvarious combinations of peptides QP20, HD20, WQ20, and SQ20.

FIG. 22. Graph showing TNFα production by PBMCs after culture withvarious combinations of peptides QP20, HD20, WQ20, and SQ20.

FIG. 23A. Graph showing IL-2 production by PBMCs from donor A afterculture with peptides QP20, HD20, WQ20, and SQ20, or CQ-22.

FIG. 23B. Graph showing IL-2 production by PBMCs from donor B afterculture with peptides QP20, HD20, WQ20, or SQ20 and combinations ofthese peptides.

FIG. 24A. Graph showing IL-17a production by PBMCs from donor A afterculture with peptides QP20, HD20, WQ20, and SQ20, or CQ-22.

FIG. 24B. Graph showing IL-17a production by PBMCs from donor B afterculture with peptides QP20, HD20, WQ20, or SQ20 and combinations ofthese peptides.

FIG. 25. Graph showing number of surface metastases in mice bearingB16-F10-LacZ tumor cells and treated with combinations of peptides.

FIG. 26. Graph showing the average number±standard deviation ofPlasmodium yoelii circumsporozoite protein (PyCS)-specific,IFNγ-secreting CD8 T cells per 0.5×10⁶ splenocytes for each cohorttested in Example 8.

FIG. 27. Graph showing the effect of the combination of QP20, HD20,WQ20, and SQ20 peptides on the mean level of serum HBsAg (hepatitis Bsurface antigen) at weeks 2 and 3 post infection.

DETAILED DESCRIPTION

This disclosure provides four peptides:

peptide amino acid sequence SEQ ID NO: QP20 QTRTVPMPKIHHPPWQNVVP 1 HD20HHHQVYQVRSHWTGMHSGHD 2 WQ20 WNLPASFHNHHIRPHEHEWIQ 3 SQ20SSYHHFKMPELHFGKNTFHQ 4

These peptides share a core sequence of HH_, which is shown above inbold, and have a strong affinity for the checkpoint receptor “programmeddeath 1” (PD-1). These peptides block the interaction of PD-1 with itsligand PD-L1 and can therefore be used to inhibit the progression of ahyperproliferative disorder, including cancer, or to treat infectiousdiseases, including persistent infections by agents such as HIV,hepatitis B virus (HBV), hepatitis C virus (HCV), and Plasmodiumfalciparum, by enhancing, stimulating, and/or increasing an individual'simmune response.

In some embodiments, any of the disclosed peptides can be modified usingchemical or recombinant methods to enhance stability or otherpharmacokinetic properties. See, e.g., US 2017/0020956. Modificationsinclude, but are not limited to, replacement of one or more L-amino acidwith its corresponding D-form, acetylation on a C- and/or N-terminalresidue, amidation on a C- and/or N-terminal residue, cyclization,esterification, glycosylation, acylation, attachment of myristic orpalmitic acid, addition of an N-terminal glycine, addition of lipophilicmoieties such as long fatty acid chains, and PEGylation.

In some embodiments, one or more of the disclosed peptides can beconjugated to various moieties, such as albumin and transthyretin, toenhance the plasma half-life of the peptide(s). Methods of preparingsuch conjugates are well known in the art (e.g., Penchala et al., 2015;Kontermann, 2016; Zorzi et al., 2017).

In some embodiments, any of the disclosed peptides can be conjugated toa partner molecule, such as a peptide or protein such as an antibodyintended to increase the half-life in vivo and/or to provide specificdelivery to a target tissue or cell. Conjugation may be direct or can bevia a linker. In some of these embodiments, the peptide can be modifiedto substitute one or more amino acids with amino acids used to attachpartner molecules, such as lysine, or by N-terminal extension of thepeptide with, e.g., 1, 2, 3, or 4 glycine spacer molecules.

Peptides, or modified versions of the peptides as described above, canbe made by any method known in the art, including synthetic methods,recombinant methods, or both. Synthetic methods include solid-phase andsolution methods, and may include the use of protective groups. See,e.g., Bodanszky et al. (1976), McOmie (1973), Merrifield (1963), Neurathet al. (1976), Stuart & Young (1984).

Recombinant production of peptides can be carried out using anynucleotide sequence(s) encoding the peptides in any suitable expressionsystem. Nucleic acid molecules encoding one or more of the disclosedpeptides can be incorporated into an expression cassette that includescontrol elements operably linked to the coding sequences. Controlelements include, but are not limited to, initiators, promoters(including inducible, repressible, and constitutive promoters),enhancers, and polyadenylation signals. Signal sequences can beincluded. The expression cassette can be provided in a vector that canbe introduced into an appropriate host cell for production of thepeptide(s). Methods of constructing expression cassettes and expressionvectors are well known. Expression vectors can include one or more ofthe expression cassettes described in the paragraphs below.

In some embodiments, an expression cassette encodes a peptide comprisingthe amino acid sequence SEQ ID NO:1. In some embodiments, an expressioncassette encodes a peptide consisting essentially of the amino acidsequence SEQ ID NO:1. In some embodiments, an expression cassetteencodes a peptide consisting of the amino acid sequence SEQ ID NO:1.

In some embodiments, an expression cassette encodes a peptide comprisingthe amino acid sequence SEQ ID NO:2. In some embodiments, an expressioncassette encodes a peptide consisting essentially of the amino acidsequence SEQ ID NO:2. In some embodiments, an expression cassetteencodes a peptide consisting of the amino acid sequence SEQ ID NO:2.

In some embodiments, an expression cassette encodes a peptide comprisingthe amino acid sequence SEQ ID NO:3. In some embodiments, an expressioncassette encodes a peptide consisting essentially of the amino acidsequence SEQ ID NO:3. In some embodiments, an expression cassetteencodes a peptide consisting of the amino acid sequence SEQ ID NO:3.

In some embodiments, an expression cassette encodes a peptide comprisingthe amino acid sequence SEQ ID NO:4. In some embodiments, an expressioncassette encodes a peptide consisting essentially of the amino acidsequence SEQ ID NO:4. In some embodiments, an expression cassetteencodes a peptide consisting of the amino acid sequence SEQ ID NO:4.

In some embodiments, an expression cassette encodes only two of thedisclosed peptides; e.g., a peptide (a) a peptide comprising, consistingessentially of, or consisting of the amino acid sequence SEQ ID NO:1 anda peptide comprising, consisting essentially of, or consisting of theamino acid sequence SEQ ID NO:2; (b) a peptide comprising, consistingessentially of, or consisting of the amino acid sequence SEQ ID NO:1 anda peptide comprising, consisting essentially of, or consisting of theamino acid sequence SEQ ID NO:3; (c) a peptide comprising, consistingessentially of, or consisting of the amino acid sequence SEQ ID NO:1 anda peptide comprising, consisting essentially of, or consisting of theamino acid sequence SEQ ID NO:4; (d) a peptide comprising, consistingessentially of, or consisting of the amino acid sequence SEQ ID NO:2 anda peptide comprising, consisting essentially of, or consisting of theamino acid sequence SEQ ID NO:3; (e) a peptide comprising, consistingessentially of, or consisting of the amino acid sequence SEQ ID NO:2 anda peptide comprising, consisting essentially of, or consisting of theamino acid sequence SEQ ID NO:4; or (f) a peptide comprising, consistingessentially of, or consisting of the amino acid sequence SEQ ID NO:3 anda peptide comprising, consisting essentially of, or consisting of theamino acid sequence SEQ ID NO:4.

In some embodiments, an expression cassette encodes only three of thedisclosed peptides; e.g., (a) a peptide comprising, consistingessentially of, or consisting of the amino acid sequence SEQ ID NO:1, apeptide comprising, consisting essentially of, or consisting of theamino acid sequence SEQ ID NO:2, and a peptide comprising, consistingessentially of, or consisting of the amino acid sequence SEQ ID NO:3;(b) a peptide comprising, consisting essentially of, or consisting ofthe amino acid sequence SEQ ID NO:1, a peptide comprising, consistingessentially of, or consisting of the amino acid sequence SEQ ID NO:2,and a peptide comprising, consisting essentially of, or consisting ofthe amino acid sequence SEQ ID NO:4; (c) a peptide comprising,consisting essentially of, or consisting of the amino acid sequence SEQID NO:2, a peptide comprising, consisting essentially of, or consistingof the amino acid sequence SEQ ID NO:3, and a peptide comprising,consisting essentially of, or consisting of the amino acid sequence SEQID NO:4; or (d) a peptide comprising, consisting essentially of, orconsisting of the amino acid sequence SEQ ID NO:1, a peptide comprising,consisting essentially of, or consisting of the amino acid sequence SEQID NO:3, and a peptide comprising, consisting essentially of, orconsisting of the amino acid sequence SEQ ID NO:4.

In some embodiments, an expression cassette encodes all four of thedisclosed peptides; i.e., a peptide comprising, consisting essentiallyof, or consisting of the amino acid sequence SEQ ID NO:1, a peptidecomprising, consisting essentially of, or consisting of the amino acidsequence SEQ ID NO:2, a peptide comprising, consisting essentially of,or consisting of the amino acid sequence SEQ ID NO:3, and a peptidecomprising, consisting essentially of, or consisting of the amino acidsequence SEQ ID NO:4.

Therapeutic Uses

The disclosed peptides have a number of therapeutic applications and canbe administered for a variety of purposes to both human and veterinarysubjects. “Administer” as used herein includes direct administration ofthe disclosed peptides or modified versions thereof as well as indirectadministration, e.g., using a nucleic acid molecule encoding thepeptides or modified versions of the peptides, as described below. Insome embodiments, administration is carried out in conjunction with oneor more other therapeutic moieties. “In conjunction with” includesadministration together with, before, or after administration of the oneor more other therapeutic moieties.

Treatment of Hyperproliferative Disorders, Including Cancer

In some embodiments, one or more of the disclosed peptides or modifiedversions thereof can be administered to inhibit the progression of ahyperproliferative disorder, such as cancer. Such inhibition mayinclude, for example, reducing proliferation of neoplastic orpre-neoplastic cells; destroying neoplastic or pre-neoplastic cells; andinhibiting metastasis or decreasing the size of a tumor.

Examples of cancers that can be treated using one or more of thedisclosed peptides or modified versions thereof include, but are notlimited to, melanomas, lymphomas, sarcomas, and cancers of the colon,kidney, stomach, bladder, brain (e.g., gliomas, glioblastomas,astrocytomas, medulloblastomas), prostate, bladder, rectum, esophagus,pancreas, liver, lung, breast, uterus, cervix, ovary, blood (e.g., acutemyeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia,chronic lymphocytic leukemia, Burkitt's lymphoma, EBV-induced B-celllymphoma).

Combination Cancer Therapies

In some embodiments, one or more of the disclosed peptides or modifiedversions thereof can be administered in conjunction with one or moretherapies or immunotherapies, such as those described below.

In some embodiments, the second therapy comprises a second agent thatreduces or blocks the activity of PD-1 (e.g., nivolumab, pembrolizumab,durvalumab) or

In some embodiments, the second therapy comprises an agent that reducesor blocks the activity of PD-L1 (e.g., atezolizumab).

In some embodiments, the second therapy comprises an agent that reducesor blocks the activity of other inhibitory checkpoint molecules and/ormolecules that suppress the immune system. These molecules include, butare not limited to:

-   -   1. Lymphocyte-activation gene-3 (LAG-3; see He et al., 2016;        Triebel et al., 1990);    -   2. cytotoxic T-lymphocyte—associated antigen 4 (CTLA-4);    -   3. V-domain Immunoglobulin Suppressor of T cell Activation        (VISTA, also known as c10orf54, PD-1H, DD1α, Gi24, Dies1, and        SISP1; see US 2017/0334990, US 2017/0112929, Gao et al., 2017,        Wang et al., 2011; Liu et al., 2015);    -   4. T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3; see        US 2017/0198041, US 2017/0029485, US 2014/0348842, Sakuishi et        al., 2010);    -   5. killer immunoglobulin-like receptors (KIRs; see US        2015/0290316);    -   6. agents that inhibit indoleamine (2,3)-dioxygenase (IDO; see        Mellemgaard et al., 2017);    -   7. B and T Lymphocyte Attenuator (BTLA; see US 2016/09222114);        and    -   8. A2A adenosine receptor (A2AR; see Beavis et al., 2015; US        2013/0267515; US 2017/0166878; Leone et al., 2015;        Mediavilla-Varela et al., 2017; Young et al., 2016).

Agents that reduce or block the activity of LAG-3 include, but are notlimited to, BMS-986016, IMP321, and GSK2831781 (He et al., 2016).

Agents that reduce or block the activity of CTLA-4 include, but are notlimited to, ipilimumab and tremelimumab.

Agents that reduce or block the activity of VISTA include, but are notlimited to, small molecules, such as CA-170, and antibodies (e.g., LeMercier et al., 2014).

Agents that reduce or block the activity of TIM-3 include, but are notlimited to, antibodies such as MBG453 and TSR-022; see Dempke et al.,2017.

Agents that reduce or block the activity of KIRs include, but are notlimited to, monoclonal antibodies such as IPH2101 and Lirilumab(BMS-986015, formerly IPH2102); see Benson & Caligiuri, 2014.

Agents that reduce or block the activity of IDO include, but are notlimited to, epacadostat and agents disclosed in US 2017/0037125.

Agents that reduce or block the activity of BTLA include, but are notlimited to, peptides (e.g., Spodzieja et al., 2017).

Agents that reduce or block the activity of A2AR include, but are notlimited to, small molecules such as CPI-444 and vipadenant.

In some embodiments, the second therapy comprises a cytokine (e.g.,interleukin 7).

In some embodiments, the second therapy comprises an agonist of astimulatory checkpoint molecule. These molecules include, but are notlimited to:

1. CD40;

2. OX40;

3. glucocorticoid-induced tumor necrosis factor-related protein (GITR);and

4. Inducible T-cell COStimulator (ICOS).

Agonists of CD40 include, but are not limited to, CD40 agonistmonoclonal antibodies such as cp-870,893, ChiLob7/4, dacetuzumab, andlucatumumab. See, e.g., Vonderheide et al., 2007; Khubchandani et al.,2009; Johnson et al., 2010; Bensinger et al., 2012; Vonderheide andGlennie, 2013; Johnson et al., 2015.

Agonists of OX40 include, but are not limited to, OX40 agonistantibodies such as MOXR0916, MED16469, MED10562, PF-045618600,GSK3174998, and INCCAGN01949, and OX40L-Fc fusion proteins, such asMEDI6383. See, e.g., Huseni et al., 2014; Linch et al., 2015;Messenheimer et al., 2017. See also Shrimali et al., 2017.

Agonists of GITR include, but are not limited to, MEDI1873. See, e.g.,Schaer et al., 2012; Tigue et al., 2017.

Agonists of ICOS include, but are not limited to, ICOS agonistantibodies JTX-2011 and GSK3359609. See, e.g., Harvey et al., 2015;Michaelson et al., 2016.

In other embodiments, the second therapy comprises a 4-1BB agonist(Shindo et al., 2015), such as urelumab; a 4-1BB antagonist (see US2017/0174773); an inhibitor of anaplastic lymphoma kinase (ALK; Wang etal., 2014; US 2017/0274074), such as crizotinib, ceritinib, alectinib,PF-06463922, NVP-TAE684, AP26113, TSR-011, X-396, CEP-37440, RXDX-101;an inhibitor of histone deacetylase (HDAC; see US 2017/0327582); a VEGFRinhibitor, such as axitinib, sunitinib, sorafenib, tivozanib,bevacizumab; and/or an anti-CD27 antibody, such as varlilumab.

In some embodiments, the second therapy comprises a cancer vaccine(e.g., Duraiswamy et al., 2013). A “cancer vaccine” is an immunogeniccomposition intended to elicit an immune response against a particularantigen in the individual to which the cancer vaccine is administered. Acancer vaccine typically contains a tumor antigen which is able toinduce or stimulate an immune response against the tumor antigen. A“tumor antigen” is an antigen that is present on the surface of a targettumor. A tumor antigen may be a molecule which is not expressed by anon-tumor cell or may be, for example, an altered version of a moleculeexpressed by a non-tumor cell (e.g., a protein that is misfolded,truncated, or otherwise mutated).

In some embodiments, the second therapy comprises a chimeric antigenreceptor (CAR) T cell therapy. See, e.g., John et al., 2013; Chong etal., 2016.

Additional Therapeutic Uses

In some embodiments, one or more of the disclosed peptides or modifiedversions thereof can be administered to treat infectious diseases,including chronic infections, caused, e.g., by viruses, fungi, bacteria,and protozoa, and helminths.

Examples of viral agents include human immunodeficiency virus (HIV),Epstein Barr Virus (EBV), Herpes simplex (HSV, including HSV1 and HSV2),Human Papillomavirus (HPV), Varicella zoster (VSV) Cytomegalovirus(CMV), and hepatitis A, B, and C viruses.

Examples of fungal agents include Aspergillus, Candida, Coccidioides,Cryptococcus, and Histoplasma capsulatum.

Examples of bacterial agents include Streptococcal bacteria (e.g.,pyogenes, agalactiae, pneumoniae), Chlamydia pneumoniae, Listeriamonocytogenes, and Mycobacterium tuberculosis.

Examples of protozoa include Sarcodina (e.g., Entamoeba), Mastigophora(e.g., Giardia), Ciliophora (e.g., Balantidium), and Sporozoa (e.g.,Plasmodium falciparum, Cryptosporidium).

Examples of helminths include Platyhelminths (e.g., trematodes,cestodes), Acanthocephalins, and Nematodes.

In some embodiments one or more of the disclosed peptides or modifiedversions thereof can be administered as a vaccine adjuvant inconjunction with a vaccine to enhance a response to vaccination (e.g.,by increasing effector T cells and/or reducing T cell exhaustion). Thevaccine can be, for example, an RNA vaccine (e.g., US 2016/0130345, US2017/0182150), a DNA vaccine, a recombinant vector, a protein vaccine,or a peptide vaccine. Such vaccines can be delivered, for example, usingvirus-like particles, as is well known in the art.

In some embodiments one or more of the disclosed peptides or modifiedversions thereof can be administered to treat sepsis.

In some embodiments one or more of the disclosed peptides or modifiedversions thereof can be administered to promote hair colorre-pigmentation. In some embodiments one or more of the disclosedpeptides or modified versions thereof can be administered to promotelightening of pigmented skin lesions.

Administration of Peptides

In some embodiments, one or more of the disclosed peptides themselves,or modified versions thereof, are administered. In some of theseembodiments, a peptide carrier system is used. A number of peptidecarrier systems are known in the art, including microparticles,polymeric nanoparticles, liposomes, solid lipid nanoparticles,hydrophilic mucoadhesive polymers, thiolated polymers, polymer matrices,nanoemulsions, and hydrogels. See Patel et al. (2014), Bruno et al.(2013), Feridooni et al. (2016). Any suitable system can be used.

In some embodiments, engineered T cell-based therapies that express andsecrete a peptide or protein can be used to deliver PD-1 inhibition atthe site of engagement of the T cell receptor with an antigen. The Tcell-based therapy could be, for example, a CAR-T cell that expressesone or more of the disclosed peptides or modified versions thereof.Either inducible or constitutive expression can be used.

In other embodiments one or more of the disclosed peptides or modifiedversions thereof are delivered using one or more nucleic acids encodingthe peptide(s) (e.g., DNA, cDNA, PNA, RNA or a combination thereof);See, e.g., US 2017/0165335. Nucleic acids encoding one or more peptidescan be delivered using a variety of delivery systems known in the art.Nucleic acid delivery systems include, but are not limited to, gene-gun;cationic lipids and cationic polymers; encapsulation in liposomes,microparticles, or microcapsules; electroporation; virus-based, andbacterial-based delivery systems. Virus-based systems include, but arenot limited to, modified viruses such as adenovirus, adeno-associatedvirus, herpes virus, retroviruses, vaccinia virus, or hybrid virusescontaining elements of one or more viruses. US 2002/0111323 describesuse of “naked DNA,” i.e., a “non-infectious, non-immunogenic,non-integrating DNA sequence,” free from “transfection-facilitatingproteins, viral particles, liposomal formulations, charged lipids andcalcium phosphate precipitating agents,” to administer a peptide.Bacterial-based delivery systems are disclosed, e.g., in Van Dessel etal. (2015) and Yang et al. (2007).

In some embodiments, a peptide is administered via an RNA moleculeencoding the peptide. In some embodiments, the RNA molecule isencapsulated in a nanoparticle. In some embodiments, the nanoparticlecomprises a cationic polymer (e.g., poly-L-lysine, polyamidoamine,polyethyleneimine, chitosan, poly(β-amino esters). In some embodiments,the nanoparticle comprises a cationic lipid or an ionizable lipid. Insome embodiments, the RNA molecule is conjugated to a bioactive ligand(e.g., N-acetylgalactosamine (GalNAc), cholesterol, vitamin E,antibodies, cell-penetrating peptides). See, e.g., Akinc et al. (2008),Akinc et al. (2009), Anderson et al. (2003), Behr (1997), Boussif et al.(1995), Chen et al. (2012), Dahlman et al. (2014), Desigaux et al.(2007), Dong et al. (2014), Dosta et al. (2015), Fenton et al. (2016),Guo et al. (2012), Howard et al. (2006), Kaczmarek et al. (2016),Kanasty et al. (2013), Kauffman et al. (2015), Kozielski et al. (2013),Leus et al. (2014), Lorenz et al. (2004), Love et al. (2010), Lynn &Langer (2000), Moschos et al. (2007), Nair et al. (2014), Nishina et al.(2008), Pack et al. (2005), Rehman et al. (2013), Schroeder et al.(2010), Tsutsumi et al. (2007), Tzeng et al. (2012), Won et al. (2009),Xia et al. (2009), Yu et al. (2016).

In some embodiments, an RNA molecule can be modified to reduce itschances of degradation or recognition by the immune system. The ribosesugar, the phosphate linkage, and/or individual bases can be modified.See, e.g., Behlke (2008), Bramsen (2009), Chiu (2003), Judge &MacLachlan (2008), Kauffman (2016), Li (2016), Morrissey (2005), Prakash(2005), Pratt & MacRae (2009), Sahin (2014), Soutschek (2004), Wittrup &Lieberman (2015). In some embodiments, the modification is one or moreof a ribo-difluorotoluyl nucleotide, a 4′-thio modified RNA, aboranophosphate linkage, a phosphorothioate linkage, a 2′-O-methyl(2′-OMe) sugar substitution, a 2′-fluoro (2′-F), a 2′-O-methoxyethyl(2′-MOE) sugar substitution, a locked nucleic acid (LNA), and an L-RNA.

Routes of Administration, Pharmaceutical Compositions, and Devices

Pharmaceutical compositions comprising an effective amount of any of theactive agents described in the paragraphs above include apharmaceutically acceptable vehicle. The “pharmaceutically acceptablevehicle” may comprise one or more substances which do not affect thebiological activity of the active agent(s) and, when administered to apatient, does not cause an adverse reaction. Pharmaceutical compositionsmay be liquid or may be lyophilized. Lyophilized compositions may beprovided in a kit with a suitable liquid, typically water for injection(WFI) for use in reconstituting the composition. Other suitable forms ofpharmaceutical compositions include suspensions, emulsions, and tablets.

Routes of administration include injection or infusion (e.g., epidural,intradermal, intramuscular, intraperitoneal, intravenous,sub-cutaneous), transdermal (e.g., US 2017/0281672), mucosal (e.g.,intranasal or oral), pulmonary, and topical (e.g., US 2017/0274010)administration. See, e.g., US 2017/0101474.

Administration can be systemic or local. In addition to local infusionsand injections, implants can be used to achieve a local administration.Examples of suitable materials include, but are not limited to,sialastic membranes, polymers, fibrous matrices, and collagen matrices.

Topical administration can be by way of a cream, ointment, lotion,transdermal patch (such as a microneedle patch), or other suitable formswell known in the art.

Administration can also be by controlled release, for example, using amicroneedle patch, pump and/or suitable polymeric materials. Examples ofsuitable materials include, but are not limited to, poly(2-hydroxy ethylmethacrylate), poly(methyl methacrylate), poly(acrylic acid),poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides(PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol),polyacrylamide, poly(ethylene glycol), polylactides (PLA),poly(lactide-co-glycolides) (PLGA), and polyorthoesters.

Devices comprising any of the active agents described above include, butare not limited to, syringes, pumps, transdermal patches, spray devices,vaginal rings, and pessaries.

Example 1. Peptide Library Screening

The TriCo-20™ (TRICO-20™) and TriCo-16™ (TRICO-16™) Phage DisplayPeptide Libraries (Creative Biolabs, 45-1 Ramsey Road, Shirley, N.Y.11967) were screened to identify binders of soluble recombinant humanPD-1 receptor. After the fourth round of panning, obvious enrichment forspecific binders was observed, and individual peptides were confirmed asweakly specific binders in a clonal phage ELISA. A fifth round ofpanning led to greater enrichment. Table 1 lists four peptides whichshowed strong specific binding in the clonal phage ELISA.

TABLE 1 Clonal Phase ELISA coated uncoated SEQ Clone signal signalpeptide sequence ID NO: QP20 0.851 0.446 QTRTVPMPKIHHPPWQNVVP 1 HD200.281 0.109 HHHQVYQVRSHWTGMHSGHD 2 WQ20 0.275 0.115WNLPASFHNHHIRPHEHEWIQ 3 SQ20 0.284 0.159 SSYHHFKMPELHFGKNTFHQ 4

Example 2. Competitive PD-1:PD-L1 Binding Inhibition Assay

Briefly, detection of cell surface PD-1 on Jurkat cells was accomplishedby incubating cells with the human PD-L1-Fc fusion protein, followed bydetection of the recombinant molecule with a fluorescently labeledanti-human Fc antibody. Flow cytometry was performed to detect bindingbetween PD-1 and the PD-L1 recombinant protein. Quantitative bindingmeasurement was then determined by mean fluorescence intensity (MFI).

Jurkat Cell-surface expression of PD1 and binding of PD-L1 to thesecells were verified as shown in FIGS. 1 and 2. The results are shown inFIGS. 3A-B, 4A-B, 5A-B, and 6A-B.

Example 3. Cell-Based Reporter Assay

A cell-based reporter assay was used to assess whether binding of thefour peptides identified above was sufficient to block the interactionwith PD-1 and its ligand PD-L1. The components of the assay include aJurkat T cell line that stably expresses human PD-1 and a luciferasereporter, a CHO cell line that stably expressed human PD-L1, and apositive control anti-PD-1 antibody that blocks the interaction of PD-1and PD-L1, resulting in a measurable effect in the assay. The luciferasereporter in the Jurkat T cell line is triggered by IL-1, NFAT, or NF-κBresponse elements in the promoter region. The Jurkat T cells arepre-treated with CD3 and immediately cryopreserved for use in the assay.Interaction of the Jurkat T cells with the PD-L1 expressing cell lineinhibits the intracellular mechanism by which the luciferase constructis activated, thereby preventing luciferase expression. A molecule thatbinds to either PD-1 on the Jurkat T cells or to PD-L1 on the CHO cellssufficiently to prevent their interaction permits the Jurkat T cells toproduce luciferase. CellTiter-Glo® (CELLTITER-GLO®, Promega) was used tomeasure luciferase expression.

The results of positive control assays using the anti-PD-1 controlantibody are shown in FIGS. 7A-B. These results demonstrate that thecontrol antibody restores luciferase expression in a dose-dependentmanner, with peak-fold inhibition of approximately 8 at an antibodyconcentration of 20 μM.

The results of assays of the peptides identified above are shown inFIGS. 8A-B. These results demonstrate that each of the four peptidesrestores luciferase expression in a dose-dependent manner, withpeak-fold inhibition of approximately 1.5 at a concentration ofapproximately 25 μM.

Example 4. Tetanus Toxoid Recall Assay Using Individual Peptides

Peptides 1-4 were tested in a human PBMC-based tetanus antigen recallassay. “Peptide CQ-22” was used as a negative control.

PBMCs were obtained from plasma of human donors and tested in vitro forrecall of tetanus toxoid. Suitable PBMCs were cryopreserved untilneeded, then thawed and cultured in a 96-wellplate. Tetanus toxoid wasadded to the cultures in the presence or absence of peptides 1-4, andthe production of cytokines and cell surface T cell activation markerswere examined.

The results of these assays are shown in FIGS. 9-15 and summarizedqualitatively in Table 2. In the table, “x” indicates no effect, “−”indicates a possible low effect, “+” indicates some effect, and “++”indicates a definite effect.

TABLE 2 peptide IL-2 IL-4 IL-6 IL-10 IL-17a IFNγ TNFα QP20 x − x x x x xHD20 − x ++ x ++ ++ ++ WQ20 − ++ ++ x ++ ++ ++ SQ20 + − ++ + ++ ++ +

The results demonstrated a trend towards modest enhancement of IL-6,IL-17α, IFNγ, and TNFα production at the highest concentrations ofpeptides. No significant enhancement of IL-2 production was detected.

Example 5. Tetanus Toxoid Recall Assay Using Combinations of Peptides

Combinations of peptides were tested in the antigen recall assaydescribed above, using a different PBMC donor and a different lot numberof tetanus toxoid. The results are shown in FIGS. 16, 17, 18, 19, 20,21, and 22. These results demonstrated that the combination of the fourpeptides combination of the four peptides QP20, HD20, WQ20, and SQ20result in increased IL-2 production and reduced IL-17a production.

The effect of peptides QP20, HD20, WQ20, and QP20 on the production ofIL-2 and IL-17a appears to be donor-specific, as shown in FIGS. 23A-Band 24A-B.

Example 6. Biacore® Assays

BIACORE® assays were carried out using a BIACORE® T-200 at 25° C. Theassay and regeneration buffers contained 10 mM HEPES (pH 7.4), 150 mMNaCl, 3 mM EDTA, and 0.05% P20. The immobilization buffer was 10 mMsodium acetate, pH 5.0. The flow rate used for immobilizing the ligandwas 5 μl/min. The flow rate for kinetics analysis was 30 μl/min.

Scouting.

12,000 response units (RU) of human and 6000 RU of mouse PD-1 receptorswere directly immobilized on flow cell 2 and flow cell 4 of the CMS chipby amine coupling method (EDC/NHS). The un-occupied sites were blockedwith 1M ethanol amine. Scouting was performed at a single analyteconcentration of 25 μM to confirm yes/no binding. Flow cell 1 was keptblank and used for reference subtraction. Binding of analyte to theligand was monitored in real time.

Full Kinetics.

Based on the scouting results, full kinetics were performed byimmobilizing higher RU of the ligand to a new chip and analyteconcentration at 25 μM, followed by serial dilution to 12.5, 6.25,3.125, 1.562, 0.78 and 0 μM concentration or as indicated. Due to faston rate and off rate, KD was determined by steady state equilibriumkinetics.

Chi square (χ2) analysis was carried out between the actual sensorgramand a sensorgram generated from the BIANALYSIS® software (black line) todetermine the accuracy of the analysis. A χ2 value within 1-2 isconsidered significant (accurate) and below 1 is highly significant(highly accurate). The results are summarized in Table 3.

TABLE 3 Ligand Rmax KA KD Conc. 10,000 RU Analyte (RU) (1/M) (M) (μM) χ²mouse PD-1 WQ-21 270 1.31 × 10³ 7.61 × 10⁻⁴ 0-25 0.0203 mouse PD-1 QP-2013.4 1.80 × 10⁴ 5.54 × 10⁻⁵ 0-25 0.0446 mouse PD-1 HD-20 76 4.25 × 10³2.35 × 10⁻⁴ 0-25 0.11 mouse PD-1 SQ-20 12.8 2.14 × 10⁴ 4.68 × 10⁻⁵ 0-250.039 human PD-1 WQ-21 84.7 3.28 × 10³ 3.05 × 10⁻⁴ 0-25 0.0309 humanPD-1 QP-20 3.83 9.36 × 10⁴ 1.07 × 10⁻⁵ 0-25 0.0569 human PD-1 HD-20 3.353.18 × 10⁵ 3.41 × 10⁻⁶  0-12.5 0.0733 human PD-1 SQ-20 4.05 1.94 × 10⁵5.16 × 10⁻⁶ 0-25 0.111 mouse PD-1 Mouse PD-L1 259 2.75 × 10⁶ 3.64 × 10⁻⁷0-50 0.105 human PD-1 Human PD-L1 213 6.92 × 10⁶ 1.44 × 10⁻⁷ 0-50 2.44

These results indicate that each of the four peptides bind both humanand mouse PD-1. QP20 and SQ20 showed the highest affinity towards mousePD-1. HD20 and SQ20 showed the highest affinity towards human PD-1.

Example 7. Experimental Metastasis Model

Efficacy of the peptides was evaluated in a B16-F10-LacZ experimentalmetastasis model. In this model, B16-F10-LacZ cells, transfected toexpress the LacZ gene that encodes β-galactoside, an intracellularenzyme, are injected into the tail vein of syngeneic mice. The cellstravel through the circulation, settle in the lungs, and form tumors.Mice are terminated 2 weeks after implant. When the enzyme cleaves itssubstrate, X-gal, the products dimerize and change color and can bedetected ex vivo. The number of metastatic tumors on the surface of thelung is then quantified by manual counting of tumors under a dissectingmicroscope.

Briefly, mice (N=7) were implanted on study day 0 with B16-F10-LacZtumor cells (5×10⁵ or 1×10⁶ cells per mouse) by intravenous injection inthe tail vein. Mice received a treatment of the peptide combination (200μg, 20 μg, or 2 μg, each peptide per dose) intravenously by tail veininjection on study days 2, 5, 7, 9 and 12. Detailed clinicalexaminations and body weights were recorded regularly during treatment.Mice were terminated on study day 14, and their lungs were removed andstained. The number of tumor metastases were counted. Treatment groupsare described in Table 4.

TABLE 4 Treatment Group N Implant Treatment Dose Route Days 1 7 5 × 10⁵QP-20, SQ-20, 200 μg IV SD 2, 5, 7, HD-20, WQ-20 9, 12 2 7 5 × 10⁵QP-20, SQ-20, 20 μg IV SD 2, 5, 7, HD-20, WQ-20 9, 12 3 7 5 × 10⁵ QP-20,SQ-20, 2 μg IV SD 2, 5, 7, HD-20, WQ-20 9, 12 4 7 5 × 10⁵ Untreated — —— 5 7 1 × 10⁶ QP-20, SQ-20, 200 μg IV SD 2, 5, 7, HD-20, WQ-20 9, 12 6 71 × 10⁶ Untreated — — —The results are shown in FIG. 25. A good dose response was observed whenmice were implanted at both cell concentrations. Mice treated with thehighest dose of peptide mixture (200 μg) had the fewest tumors (average97), and mice treated with the lowest dose of peptide mixture (2 μg) hadthe most tumors (average 205). Similarly, in the two groups that wereimplanted with high tumor numbers, the untreated group had significantlymore tumors. This indicates that the 4 peptides in combination showed adose-dependent efficacy on B16-F10-LacZ tumor growth in vivo. Moreover,the peptide combination was well tolerated by the mice and did not haveany acute adverse effects on animal health.

Example 8. Effect of Peptide Combination on the Immunogenicity of aMalaria Vaccine

Immunogenicity of the peptide combination as a prophylactic vaccineadjuvant was assessed in a mouse model of malaria. Balb/c mice immunizedwith an adenovirus-based malaria vaccine expressing the Plasmodiumyoelli circumsporozoite protein (AdPyCS) were given 200 μg of thepeptide combination, anti-PD-1 mAb, anti-PDL1 mAb, or the negativecontrol peptide ovalbumin (OVA) on days 1, 3, 5, and 7 afterimmunization with AdPyCS (Table 5). Note that no additional adjuvant wasadded to the AdPyCS antigen. Spleens were collected 12 days afterimmunization, and the number of splenic PyCS-specific, IFNγ-secretingCD8⁺ T cells was determined via ELISpot assay. Note that for the ELISpotassay, splenocytes were stimulated with the SYVPSAEQI peptide (SEQ IDNO:5), an H-2Kd-restricted CD8⁺ T cell epitope of PyCS.

TABLE 5 Cohort Test Sample # Mice Route Treatment days 1 AdPyCS only 5 —— 2 AdPyCS + control OVA 5 i.p. 0, 1, 3, 5, 7 peptide (200 μg) 3AdPyCS + peptide combo 5 i.p. 0, 1, 3, 5, 7 (200 μg) 4 AdPyCS +anti-PD-1 antibody 5 i.p. 0, 1, 3, 5, 7 (200 μg) 5 AdPyCS + anti-PDL1 5i.p. 0, 1, 3, 5, 7 antibody (200 μg)

FIG. 26 shows the average number±standard deviation of CSP-specific,IFNγ-secreting CD8⁺ T cells per 0.5×10⁶ splenocytes for each cohort.Significant differences in the average number±standard deviation ofCSP-specific, IFNγ-secreting CD8⁺ T cells per 0.5×10⁶ splenocytesbetween the AdPyCS alone (Cohort 1) and the peptide combination (Cohort3), anti-PD-1 antibody (Cohort 4) or anti-PD-L1 antibody (Cohort 5) weredetected using the one-way ANOVA test (***p<0.001, and *p<0.05). Theseresults demonstrate that the peptide combination (Cohort 3) isfunctionally active in vivo, increasing the number of CSP-specific,IFNγ-secreting CD8⁺ T cells ˜1.6-fold relative to AdPyCS alone (Cohort1), which was similar to changes with anti-PD-1 or -PD-L1 antibody(Cohort 4 and 5).

Example 9. Effect of Peptide Combination on Survival in a Model ofSepsis

Sepsis can negatively alter T cell function and survival, however thiscan be reversed when the PD-1:PDL1 interaction is blocked, which resultsin improved survival. Thus the efficacy of the peptide combination wasassessed in a representative, clinically relevant model of sepsis whereCD1 mice are subjected to cecal ligation and puncture (CLP) to induceintra-abdominal peritonitis. For this study, 200 μg of either thepeptide combination or anti-PD-1 antibody were administered i.v. at 2,24, 48, 72 and 96 hours after surgery. A vehicle control group was alsoincluded. Six mice were in each group. All mice were checked twice dailyfor signs of morbidity and mortality. Administration of the peptidecombination conferred an enhanced survival advantage over the vehiclecontrol group where the peptide combination showed a 2-fold highersurvival rate (Table 6). Moreover, survival in the peptide combinationgroup was slightly above treatment with anti-PD-1 antibody.

TABLE 6 Group % Survival Vehicle Control 50% Anti-PD-1 antibody 83% PD-1Peptide Combo 100%

Example 10. Effect of Peptide Combination on Serum HBsAg Levels inHBV-Infected Mice

The combination of QP20, HD20, WQ20, and SQ20 peptides was assessed in ahepatitis B virus (HBV) mouse model where the role of PD-1 in T cellexhaustion and immunotolerance is documented (Tzeng et al., 2012; Ye etal., 2015). PD-1 is elevated in the hepatic T cells of mice withpersistent HBV infection but not in animals that have cleared theinfection. In this model, it has been shown that inhibition of thePD-1/PD-L1 interaction with an anti-PD-1 mAb both increasesantigen-specific IFNγ production by hepatic T cells and reverses HBVpersistence (Tzeng et al., 2012). This mouse model of persistent HBVpresented an opportunity to test whether the combination of QP20, HD20,WQ20, and SQ20 peptides can reverse T cell exhaustion in vivo and aidthe immune system in controlling viral infection.

Mice infected with HBV were treated with saline (negative control), 200μg of QP20, HD20, WQ20, and SQ20 peptides combined, or 200 μg anti-PD-1mAb at 9 time points, 2 days prior to infection and days 1, 3, 6, 9, 12,14, 17 and 20 post infection. The level of serum HB surface antigen(HBsAg) was monitored by ELISA on days 7, 14, and 21 to follow theinfection (higher levels of serum HBsAg are reflective of higher viraltiter) and detect the immune enhancement activity of the combination ofQP20, HD20, WQ20, and SQ20 peptides. The group treated with thecombination of QP20, HD20, WQ20, and SQ20 peptides showed significantlylower mean level of serum HBsAg at weeks 2 and 3 post infection (p<0.05,1-way ANOVA, Tukey's Multiple Comparison Test) compared to the salinenegative control (FIG. 27).

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1. A method of inhibiting the progression of a hyperproliferativedisorder, treating an infectious disease, enhancing a response tovaccination, treating sepsis, promoting hair re-pigmentation, orpromoting lightening of a pigmented skin lesion, comprisingadministering to an individual in need thereof an effective amount of atleast one peptide selected from the group consisting of (i) a peptidecomprising the amino acid sequence SEQ ID NO:1; (ii) a peptidecomprising the amino acid sequence SEQ ID NO:2; (iii) a peptidecomprising the amino acid sequence SEQ ID NO:3; and (iv) a peptidecomprising the amino acid sequence SEQ ID NO:4, wherein theadministration comprises: (a) administration of a nucleic acid encodingthe at least one peptide; (b) administration of a peptide carrier systemcomprising the at least one peptide; or (c) administration of a CAR-Tcell that expresses the at least one peptide.
 2. The method of claim 1,which comprises administration of the nucleic acid encoding the at leastone peptide, wherein the nucleic acid is selected from the groupconsisting of DNA, cDNA, PNA, and RNA.
 3. The method of claim 1, whichcomprises the administration of the peptide carrier system comprisingthe at least one peptide, wherein the peptide carrier system is selectedfrom the group consisting of a microparticle, a polymeric nanoparticle,a liposome, a solid lipid nanoparticle, a hydrophilic mucoadhesivepolymer, a thiolated polymer, a polymer matrix, a nanoemulsion, and ahydrogel.
 4. The method of claim 1, wherein the at least one peptide isadministered to inhibit progression of the hyperproliferative disorder.5. The method of claim 4, wherein the hyperproliferative disorder is acancer.
 6. The method of claim 5, wherein the cancer is a melanoma. 7.The method of claim 5, further comprising administering a second therapyto the patient.
 8. The method of claim 7, wherein the second therapycomprises a cancer vaccine.
 9. The method of claim 7, wherein the secondtherapy comprises a chimeric antigen receptor (CAR) T cell therapy. 10.The method of claim 7, wherein the second therapy comprises reducing orblocking activity of a molecule selected from the group consisting ofPD-1, PD-L1, lymphocyte-activation gene-3 (LAG-3), cytotoxicT-lymphocyte—associated antigen 4 (CTLA-4), V-domain ImmunoglobulinSuppressor of T cell Activation (VISTA), T-cell Immunoglobulin domainand Mucin domain 3 (TIM-3), a killer immunoglobulin-like receptor (KIR),indoleamine (2,3)-dioxygenase (IDO), B and T Lymphocyte Attenuator(BTLA), A2A adenosine receptor (A2AR).
 11. The method of claim 7,wherein the second therapy comprises a cytokine.
 12. The method of claim7, wherein the second therapy comprises an agonist of a moleculeselected from the group consisting of CD40, OX40, glucocorticoid-inducedtumor necrosis factor-related protein (GITR), and Inducible T-cellCOStimulator (ICOS).
 13. The method of claim 7, wherein the secondtherapy comprises a therapeutic agent selected from the group consistingof a 4-1BB agonist, a 4-1BB antagonist, an inhibitor of anaplasticlymphoma kinase (ALK), an inhibitor of histone deacetylase (HDAC), andan inhibitor of VEGFR.
 14. The method of claim 1, wherein the at leastone peptide is administered to treat an infectious disease.
 15. Themethod of claim 14, wherein the infectious disease is malaria orhepatitis B.
 16. The method of claim 14, wherein the at least onepeptide is administered as a vaccine adjuvant to a vaccine against theinfectious disease.
 17. The method of claim 1, wherein the at least onepeptide is administered to treat sepsis.
 18. The method of claim 1,wherein the at least one peptide is administered to promote hairre-pigmentation or to promote lightening of a pigmented skin lesion. 19.An expression construct encoding up to four peptides selected from thegroup consisting of (i) a peptide consisting of the amino acid sequenceSEQ ID NO:1; (ii) a peptide consisting of the amino acid sequence SEQ IDNO:2; (iii) a peptide consisting of the amino acid sequence SEQ ID NO:3;and (iv) a peptide consisting of the amino acid sequence SEQ ID NO:4.20. An RNA molecule encoding up to four peptides selected from the groupconsisting of (i) a peptide consisting of the amino acid sequence SEQ IDNO:1; (ii) a peptide consisting of the amino acid sequence SEQ ID NO:2;(iii) a peptide consisting of the amino acid sequence SEQ ID NO:3; and(iv) a peptide consisting of the amino acid sequence SEQ ID NO:4 andcomprising at least one modification selected from the group consistingof (i) modification of a ribose sugar, (ii) modification of a phosphatelinkage, and (iii) modification of a base.
 21. The RNA molecule of claim20, wherein the at least one modification is selected from the groupconsisting of a ribo-difluorotoluyl nucleotide, a 4′-thio modified RNA,a boranophosphate linkage, a phosphorothioate linkage, a 2′-O-methyl(2′-OMe) sugar substitution, a 2′-fluoro (2′-F), a 2′-O-methoxyethyl(2′-MOE) sugar substitution, a locked nucleic acid (LNA), and an L-RNA.22. A pharmaceutical composition comprising: (a) a nucleic acid moleculeencoding up to four peptides selected from the group consisting of (i) apeptide consisting of the amino acid sequence SEQ ID NO:1; (ii) apeptide consisting of the amino acid sequence SEQ ID NO:2; (iii) apeptide consisting of the amino acid sequence SEQ ID NO:3; and (iv) apeptide consisting of the amino acid sequence SEQ ID NO:4; and (b) apharmaceutically acceptable carrier.
 23. The pharmaceutical compositionof claim 22, wherein the nucleic acid molecule is an RNA molecule.